Project Summary The transition from dependence on maternal transcripts deposited into the egg to newly transcribed zygotic transcripts is carefully regulated to ensure proper development of early embryos. To provide insight into the regulation of zygotic gene expression timing, many recent studies have focused on how maternal transcription factors activate the zygotic genome in Drosophila early embryos. On the other hand, the mechanism of action of ubiquitously expressed repressors is not well understood, though recent studies have shown they also impact expression of spatially-localized genes. The experiments proposed here will investigate a role for ubiquitous repressors in controlling enhancer action. Specifically, we propose that sequence-specific, broadly-expressed repressors act as guardians of zygotic genome activation in early embryos, controlling the action of particular enhancers to regulate timing of gene expression, and thus effectively manage the maternal-zygotic transition. Repressors of transcription are understudied compared to activators, yet we propose both types of transcriptional regulators are equally important. We hypothesize that the decision of whether or not a cis-regulatory element functions to support gene expression depends not only on the amount of activation but also on how much repression must be overcome. To provide insight into broadly-expressed repressors? mechanism(s) of action, our experimental approach has two specific aims. Aim 1. Investigate whether chromatin accessibility is regulated by broadly-expressed repressors. We will use Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) to investigate whether changes in repressor levels affect chromatin accessibility. This method allows assay of single embryos that have been carefully staged to provide a time-course of chromatin accessibility in the early embryo. The goal of our experiments is to increase or decrease repressor levels, either by assaying mutants or introducing targeted mutations into enhancer sequences, to investigate a role for these factors in regulating chromatin opening. Aim 2. Test the idea that broadly-expressed repressors regulate Bicoid-dependent morphogen outputs. Bicoid is a maternally deposited transcription factor that regulates gene expression in a concentration-dependent manner. Rather than transcriptional activation being linked directly to absolute Bicoid concentration, broadly-expressed repressors may act to modulate the morphogen effective concentration: for example, higher levels of Bicoid may be required to activate enhancers bound by repressors compared to those not bound. We will assay for genetic interaction between activators and repressors and also use chromatin-immunoprecipitation (ChIP) to test whether repressors influence Bicoid?s DNA-binding in vivo. The insights gained here regarding the role of broadly-expressed repressors will likely be applicable to higher organisms, including vertebrates, as cis-regulatory mechanisms are generally conserved in metazoan animals. ?? ? ?? ? ?